Abstract Background: The human upper digestive tract microbial community (microbiota) is not well characterized and few studies have explored how it relates to human health. We examined the relationship between upper digestive tract microbiota and two cancer-predisposing states, serum pepsinogen I/pepsinogen II ratio (PGI/II; predictor of gastric cancer risk) and esophageal squamous dysplasia (ESD; the precursor lesion of esophageal squamous cell carcinoma; ESCC) in a cross-sectional design. Methods: The Human Oral Microbe Identification Microarray was used to test for the presence of 272 bacterial species in 333 upper digestive tract samples from a Chinese cancer screening cohort. Serum PGI and PGII were determined by ELISA. ESD was determined by chromoendoscopy with biopsy. Results: Lower microbial richness (number of bacterial genera per sample) was significantly associated with lower PGI/II ratio (P = 0.034) and the presence of ESD (P = 0.018). We conducted principal component (PC) analysis on a β-diversity matrix (pairwise difference in microbiota), and observed significant correlations between PC1, PC3, and PGI/II (P = 0.004 and 0.009, respectively), and between PC1 and ESD (P = 0.003). Conclusions: Lower microbial richness in upper digestive tract was independently associated with both cancer-predisposing states in the esophagus and stomach (presence of ESD and lower PGI/II). Impact: These novel findings suggest that the upper digestive tract microbiota may play a role in the etiology of chronic atrophic gastritis and ESD, and therefore in the development of gastric and esophageal cancers. Cancer Epidemiol Biomarkers Prev; 23(5); 735–41. ©2014 AACR.